Esterases

ABSTRACT

Esterase enzymes derived from various Staphylothermus, Pyrodictium, Archaeoglobus, Aquifex, M11TL, Thermococcus, Teredinibacter and Sulfolobus organisms are disclosed. The enzymes are produced from native or recombinant host cells and can be utilized in pharmaceutical, agricultural and other industries.

RELATED APPLICATIONS

[0001] This application is a divisional of co-pending U. S. patentapplication Ser. No. 08/602,359, filed Feb. 17, 1996.

SPECIFICATION

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production andisolation of such polynucleotides and polypeptides. More particularly,the polynucleotides and polypeptides of the present invention have beenputatively identified as esterases. Esterases are enzymes that catalyzethe hydrolysis of ester groups to organic acids and alcohols.

[0003] Many esterases are known and have been discovered in a broadvariety of organisms, including bacteria, yeast and higher animals andplants. A principal example of esterases are the lipases, which are usedin the hydrolysis of lipids, acidolysis(replacement of an esterifiedfatty acid with a free fatty acid) reactions,transesterification(exchange of fatty acids betweentriglycerides)reactions, and in ester synthesis. The major industrialapplications for lipases include: the detergent industry, where they areemployed to decompose fatty materials in laundry stains into easilyremovable hydrophilic substances; the food and beverage industry wherethey are used in the manufacture of cheese, the ripening and flavoringof cheese, as antistaling agents for bakery products, and in theproduction of margarine and other spreads with natural butter flavors;in waste systems; and in the pharmaceutical industry where they are usedas digestive aids.

[0004] The polynucleotides and polypeptides of the present inventionhave been identified as esterases as a result of their enzymaticactivity.

[0005] In accordance with one aspect of the present invention, there areprovided novel enzymes, as well as active fragments, analogs andderivatives thereof.

[0006] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding the enzymes of thepresent invention including mRNAs, cDNAs, genomic DNAs as well as activeanalogs and fragments of such enzymes.

[0007] In accordance with another aspect of the present invention thereare provided isolated nucleic acid molecules encoding maturepolypeptides expressed by the DNA contained in ATCC Deposit No.______.

[0008] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptides byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a nucleic acid sequence of thepresent invention, under conditions promoting expression of said enzymesand subsequent recovery of said enzymes.

[0009] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such enzymes, orpolynucleotides encoding such enzymes for hydrolyzing ester groups toyield an organic acid and an alcohol. The esterases of the invention arestable at high temperatures and in organic solvents and, thus, aresuperior for use in production of optically pure chiral compounds usedin pharmaceutical, agricultural and other chemical industries.

[0010] In accordance with yet a further aspect of the present invention,there are also provided nucleic acid probes comprising nucleic acidmolecules of sufficient length to hybridize to a nucleic acid sequenceof the present invention.

[0011] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such enzymes, orpolynucleotides encoding such enzymes, for in vitro purposes related toscientific research, for example, to generate probes for identifyingsimilar sequences which might encode similar enzymes from otherorganisms by using certain regions, i.e., conserved sequence regions, ofthe nucleotide sequence.

[0012] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0013] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0014]FIG. 1 is an illustration of the full-length DNA (SEQ ID NO:23)and corresponding deduced amino acid sequence (SEQ ID NO:33) ofStaphylothermus marinus F1-12LC of the present invention. Sequencing wasperformed using a 378 automated DNA sequencer (Applied Biosystems, Inc.)for all sequences of the present invention.

[0015]FIG. 2 is an illustration of the full-length DNA (SEQ ID NO:24)and corresponding deduced amino acid sequence (SEQ ID NO:34) ofPyrodictium TAG11-17LC.

[0016]FIG. 3 is an illustration of the full-length DNA (SEQ ID NO:25)and corresponding deduced amino acid sequence (SEQ ID NO:35) ofArchaeoglobus venificus SNP6-24LC.

[0017]FIG. 4 is an illustration of the full-length DNA (SEQ ID NO:26)and corresponding deduced amino acid sequence (SEQ ID NO:36) of Aquifexpyrophilus-28LC.

[0018]FIG. 5 is an illustration of the full-length DNA (SEQ ID NO:27)and corresponding deduced amino acid sequence (SEQ ID NO:37) ofM11TL-29L.

[0019]FIG. 6 is an illustration of the full-length DNA (SEQ ID NO:28)and corresponding deduced amino acid sequence (SEQ ID NO:38) ofThermococcus CL-2-30LC.

[0020]FIG. 7 is an illustration of the full-length DNA (SEQ ID NO:29)and corresponding deduced amino acid sequence (SEQ ID NO:39) of AquifexVF5-34LC.

[0021]FIG. 8 is an illustration of the full-length DNA (SEQ ID NO:30)and corresponding deduced amino acid sequence (SEQ ID NO:40) ofTeredinibacter-42L.

[0022]FIG. 9 is an illustration of the full-length DNA (SEQ ID NO:31)and corresponding deduced amino acid sequence (SEQ ID NO:41) ofArchaeoglobus fulgidus VC16-16MC.

[0023]FIG. 10 is an illustration of the full-length DNA (SEQ ID NO:32)and corresponding deduced amino acid sequence (SEQ ID NO:42) ofSurfolobus solfataricus P1-8LC.

[0024] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0025] A coding sequence is “Operably linked to” another coding sequencewhen RNA polymerase will transcribe the two coding sequences into asingle mRNA, which is then translated into a single polypeptide havingamino acids derived from both coding sequences. The coding sequencesneed not be contiguous to one another so long as the expressed sequencesultimately process to produce the desired protein.

[0026] “Recombinant” enzymes refer to enzymes produced by recombinantDNA techniques; i.e., produced from cells transformed by an exogenousDNA construct encoding the desired enzyme. “Synthetic” enzymes are thoseprepared by chemical synthesis.

[0027] A DNA “coding sequence of” or a “nucleotide sequence encoding” aparticular enzyme, is a DNA sequence which is transcribed and translatedinto an enzyme when placed under the control of appropriate regulatorysequences.

[0028] In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode for themature enzymes having the deduced amino acid sequences of FIGS. 1-10(SEQ ID NOS:23-32).

[0029] In accordance with another aspect of the present invention, thereare provided isolated polynucleotides encoding the enzymes of thepresent invention. The deposited material is a mixture of genomic clonescomprising DNA encoding an enzyme of the present invention. Each genomicclone comprising the respective DNA has been inserted into a pBluescriptvector (Stratagene, La Jolla, Calif.). The deposit has been depositedwith the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. 20852, USA, on Dec. 13, 1995 and assigned ATCC DepositNo.______.

[0030] The deposit(s) have been made under the terms of the BudapestTreaty on the International Recognition of the deposit ofmicro-organisms for purposes of patent procedure. The strains will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. These deposits are provided merely asconvenience to those of skill in the art and are not an admission that adeposit would be required under 35 U.S.C. §112. The sequences of thepolynucleotides contained in the deposited materials, as well as theamino acid sequences of the polypeptides encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0031] The polynucleotides of this invention were originally recoveredfrom genomic gene libraries derived from the following organisms:

[0032]Staphylothermus marinus F1 is a thermophilic sulfur archaea whichwas isolated in Vulcano, Italy. It grows optimally at 85° C.(T_(max)=98° C.) at pH 6.5.

[0033] Pyrodictium TAG11 is a thermophilic sulfur archaea which wasisolated in the Middle Atlantic Ridge. It grows optimally at 103° C.(T_(max)=110° C.) at pH 6.5.

[0034]Archaeoglobus venificus SNP6 was isolated in the Middle AtlanticRidge and grows optimally at 75° C. (T_(max)=92° C.) at pH 6.9.

[0035]Aquifex pyrophilus K01 5a was isolated at Kolbeinsey Ridge, Northof Iceland. This marine organism is a gram-negative, rod-shaped,strictly chemolithoautrophic, knall gas bacterium. It grows optimally at85° C. (T_(max)=950° C.) at pH 6.8.

[0036] M11TL is a new species of Desulfurococcus which was isolated fromDiamond Pool (formerly Jim's Black Pool) in Yellowstone. The organismgrows heterotrophically by fermentation of different organic materials(sulfur is not necessary) in grape-like aggregates optimally at 85-88°C. in a low salt medium at pH 7.0.

[0037] Thermococcus CL-2 was isolated in the North Cleft Segment of theJuan de Fuca Ridge from a severed alvinellid worm residing on a “blacksmoker” sulfide structure. This marine archaea forms pleomorphic cocci,and grows optimally at 88° C.

[0038] Aquifex VF5 was isolated at a beach in Vulcano, Italy. Thismarine organism is a gram-negative, rod-shaped, strictlychemolithoautotrophic, knall gas bacterium. It grows optimally at 85° C.(T_(max)=95° C.) at pH 6.8.

[0039] Teredinibacter (pure) is an endosymbiont of the shipworm Bankiagouldi. The organism has straight to slightly bent 5-10 μm rods, andforms spiral cells as stationary phase is met. The organism wasdescribed in Science (1983) 22:1401-1403. It grows optimally at 30° C.at pH 8.0.

[0040]Archaeoglobus fulgidus VC16 was isolated in Vulcano, Italy. Theorganism grows optimally at 85° C. (T_(max)=92° C.) at pH 7.0.

[0041]Sutfolobus solfataricus P1 grows optimally at 85° C. (T_(max)=87°C.) at pH 2.0.

[0042] Accordingly, the polynucleotides and enzymes encoded thereby areidentified by the organism from which they were isolated, and aresometimes hereinafter referred to as F1/12LC (FIG. 1 and SEQ ID NOS:23and 33), TAG11/17LC (FIG. 2 and SEQ ID NOS:24 and 34), SNP6/24LC (FIG. 3and SEQ ID NOS:25 and 35), AqP/28LC (FIG. 4 and SEQ ID NOS:26 and 36),M11TL/29L (FIG. 5 and SEQ ID NOS:27 and 37), CL-2/30LC (FIG. 6 and SEQID NOS:28 and 38), VF5/34LC (FIG. 7 and SEQ ID NOS:29 and 39), Trb/42L(FIG. 8 and SEQ ID NOS:30 and 40), VC16/16MC (FIG. 9 and SEQ ID NOS:31and 41) and P1/8LC (FIG. 10 and SEQ ID NOS: 32 and 42).

[0043] The polynucleotides and polypeptides of the present inventionshow identity at the nucleotide and protein level to known genes andproteins encoded thereby as shown in Table 1. TABLE 1 Protein ProteinDNA Gene w/closest Similarity Identity Identity Enzyme Homology(Organism) (%) (%) (%) F1/12LC No significant homology — — — TAG11/17LCNo significant homology — — — SNP6/24LC PIR S34609 - 46 27 42carboxylesterase Pseudomones sp. (strain KWI-56) open reading frame ofunknown function in E. coli. AqP/29LC 53 31 38 M11TL/29LC No significanthomology — — — CL02/30LC No significant homology — — — VF5/34LCIdentified by homology 84 71 71 to 28LC; also homologous to ORF ofunknown function 5′ of tgs in E. coli Trb/42L No significant homology —— — P1-8LC VC16-16MC

[0044] All the clones identified in Table 1 encode polypeptides whichhave esterase activity.

[0045] This invention, in addition to the isolated nucleic acidmolecules encoding the enzymes of the present invention, also providessubstantially similar sequences. Isolated nucleic acid sequences aresubstantially similar if: (i) they are capable of hybridizing underconditions hereinafter described, to the polynucleotides of SEQ IDNOS:23-32; (ii) or they encode DNA sequences which are degenerate to thepolynucleotides of SEQ ID NOS:23-32. Degenerate DNA sequences encode theamino acid sequences of SEQ ID NOS:33-42, but have variations in thenucleotide coding sequences. As used herein, substantially similarrefers to the sequences having similar identity to the sequences of theinstant invention. The nucleotide sequences that are substantially thesame can be identified by hybridization or by sequence comparison.Enzyme sequences that are substantially the same can be identified byone or more of the following: proteolytic digestion, gel electrophoresisand/or microsequencing.

[0046] One means for isolating the nucleic acid molecules encoding theenzymes of the present invention is to probe a gene library with anatural or artificially designed probe using art recognized procedures(see, for example: Current Protocols in Molecular Biology, Ausubel F. M.et al. (EDS.) Green Publishing Company Assoc. and John WileyInterscience, New York, 1989, 1992). It is appreciated by one skilled inthe art that the polynucleotides of SEQ ID NOS:23-32, or fragmentsthereof (comprising at least 12 contiguous nucleotides), areparticularly useful probes. Other particularly useful probes for thispurpose are hybridizable fragments of the sequences of SEQ ID NOS:1-22(i.e., comprising at least 12 contiguous nucleotides).

[0047] With respect to nucleic acid sequences which hybridize tospecific nucleic acid sequences disclosed herein, hybridization may becarried out under conditions of reduced stringency, medium stringency oreven stringent conditions. As an example of oligonucleotidehybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/mL polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at Tm ⁻10° C. for the oligo-nucleotide probe. The membrane is thenexposed to auto-radiographic film for detection of hybridizationsignals.

[0048] Stringent conditions means hybridization will occur only if thereis at least 90% identity, preferably at least 95% identity and mostpreferably at least 97% identity between the sequences. See J. Sambrooket al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold SpringHarbor Laboratory (1989) which is hereby incorporated by reference inits entirety.

[0049] As used herein, a first DNA (RNA) sequence is at least 70% andpreferably at least 80% identical to another DNA (RNA) sequence if thereis at least 70% and preferably at lest a 80% or 90% identity,respectively, between the bases of the first sequence and the bases ofthe another sequence, when properly aligned with each other, for examplewhen aligned by BLASTN.

[0050] The present invention relates to polynucleotides which differfrom the reference polynucleotide such that the changes are silentchanges, for example the change do not alter the amino acid sequenceencoded by the polynucleotide. The present invention also relates tonucleotide changes which result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference polynucleotide. In a preferred aspect of the invention thesepolypeptides retain the same biological action as the polypeptideencoded by the reference polynucleotide.

[0051] The polynucleotides of this invention were recovered from genomicgene libraries from the organisms listed in Table 1. Gene libraries weregenerated in the Lambda ZAP II cloning vector (Stratagene CloningSystems). Mass excisions were performed on these libraries to generatelibraries in the pBluescript phagemid. Libraries were generated andexcisions were performed according to the protocols/methods hereinafterdescribed.

[0052] The polynucleotides of the present invention may be in the formof RNA or DNA which DNA includes cDNA, genomic DNA, and synthetic DNA.The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequences which encodes the mature enzymes may be identical tothe coding sequences shown in FIGS. 1-10 (SEQ ID NOS:23-32) or may be adifferent coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same matureenzymes as the DNA of FIGS. 1-10 (SEQ ID NOS:23-32).

[0053] The polynucleotide which encodes for the mature enzyme of FIGS.1-10 (SEQ ID NOS:33-42) may include, but is not limited to: only thecoding sequence for the mature enzyme; the coding sequence for themature enzyme and additional coding sequence such as a leader sequenceor a proprotein sequence; the coding sequence for the mature enzyme (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature enzyme.

[0054] Thus, the term “polynucleotide encoding an enzyme (protein)”encompasses a polynucleotide which includes only coding sequence for theenzyme as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0055] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the enzymes having the deduced amino acidsequences of FIGS. 1-10 (SEQ ID NOS:33-42). The variant of thepolynucleotide may be a naturally occurring allelic variant of thepolynucleotide or a non-naturally occurring variant of thepolynucleotide.

[0056] Thus, the present invention includes polynucleotides encoding thesame mature enzymes as shown in FIGS. 1-10 (SEQ ID NOS:23-32) as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the enzymes of FIGS. 1-10 (SEQ ID NOS:23-32).Such nucleotide variants include deletion variants, substitutionvariants and addition or insertion variants.

[0057] As hereinabove indicated, the polynucleotides may have a codingsequence which is a naturally occurring allelic variant of the codingsequences shown in FIGS. 1-10 (SEQ ID NOS:23-32). As known in the art,an allelic variant is an alternate form of a polynucleotide sequencewhich may have a substitution, deletion or addition of one or morenucleotides, which does not substantially alter the function of theencoded enzyme.

[0058] Fragments of the full length gene of the present invention may beused as hybridization probes for a cDNA or a genomic library to isolatethe full length DNA and to isolate other DNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 10, preferably at least 15, and even morepreferably at least 30 bases and may contain, for example, at least 50or more bases. The probe may also be used to identify a DNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of genomic DNA to determine which members ofthe library the probe hybridizes to.

[0059] It is also appreciated that such probes can be and are preferablylabeled with an analytically detectable reagent to facilitateidentification of the probe. Useful reagents include but are not limitedto radioactivity, fluorescent dyes or enzymes capable of catalyzing theformation of a detectable product. The probes are thus useful to isolatecomplementary copies of DNA from other sources or to screen such sourcesfor related sequences.

[0060] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode enzymes which eitherretain substantially the same biological function or activity as themature enzyme encoded by the DNA of FIGS. 1-10 (SEQ ID NOS:23-32).

[0061] Alternatively, the polynucleotide may have at least 15 bases,preferably at least 30 bases, and more preferably at least 50 baseswhich hybridize to any part of a polynucleotide of the present inventionand which has an identity thereto, as hereinabove described, and whichmay or may not retain activity. For example, such polynucleotides may beemployed as probes for the polynucleotides of SEQ ID NOS:23-32, forexample, for recovery of the polynucleotide or as a diagnostic probe oras a PCR primer.

[0062] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% identity and morepreferably at least a 95% identity to a polynucleotide which encodes theenzymes of SEQ ID NOS:33-42 as well as fragments thereof, whichfragments have at least 15 bases, preferably at least 30 bases and mostpreferably at least 50 bases, which fragments are at least 90%identical, preferably at least 95% identical and most preferably atleast 97% identical under stringent conditions to any portion of apolynucleotide of the present invention.

[0063] The present invention further relates to enzymes which have thededuced amino acid sequences of FIGS. 1-10 (SEQ ID NOS:23-32) as well asfragments, analogs and derivatives of such enzyme.

[0064] The terms “fragment,” “derivative” and “analog” when referring tothe enzymes of FIGS. 1-10 (SEQ ID NOS:33-42) mean enzymes which retainessentially the same biological function or activity as such enzymes.Thus, an analog includes a proprotein which can be activated by cleavageof the proprotein portion to produce an active mature enzyme.

[0065] The enzymes of the present invention may be a recombinant enzyme,a natural enzyme or a synthetic enzyme, preferably a recombinant enzyme.

[0066] The fragment, derivative or analog of the enzymes of FIGS. 1-10(SEQ ID NOS:33-42) may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature enzyme is fused withanother compound, such as a compound to increase the half-life of theenzyme (for example, polyethylene glycol), or (iv) one in which theadditional amino acids are fused to the mature enzyme, such as a leaderor secretory sequence or a sequence which is employed for purificationof the mature enzyme or a proprotein sequence. Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

[0067] The enzymes and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0068] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or enzymepresent in a living animal is not isolated, but the same polynucleotideor enzyme, separated from some or all of the coexisting materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or enzymes could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

[0069] The enzymes of the present invention include the enzymes of SEQID NOS:33-42 (in particular the mature enzyme) as well as enzymes whichhave at least 70% similarity (preferably at least 70% identity) to theenzymes of SEQ ID NOS:33-42 and more preferably at least 90% similarity(more preferably at least 90% identity) to the enzymes of SEQ IDNOS:33-42 and still more preferably at least 95% similarity (still morepreferably at least 95% identity) to the enzymes of SEQ ID NOS:33-42 andalso include portions of such enzymes with such portion of the enzymegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

[0070] As known in the art “similarity” between two enzymes isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one enzyme to the sequence of a second enzyme.

[0071] A variant, i.e. a “fragment”, “analog” or “derivative”polypeptide, and reference polypeptide may differ in amino acid sequenceby one or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

[0072] Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

[0073] Most highly preferred are variants which retain the samebiological function and activity as the reference polypeptide from whichit varies.

[0074] Fragments or portions of the enzymes of the present invention maybe employed for producing the corresponding full-length enzyme bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length enzymes. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

[0075] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof enzymes of the invention by recombinant techniques.

[0076] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0077] The polynucleotides of the present invention may be employed forproducing enzymes by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing an enzyme. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0078] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0079] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0080] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0081] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0082] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Bacillussubtilis; fungal cells, such as yeast; insect cells such as DrosophilaS2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

[0083] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBluescript II KS, ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia);Eukaryotic: pXT1, pSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVL, SV40(Pharmacia). However, any other plasmid or vector may be used as long asthey are replicable and viable in the host.

[0084] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0085] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0086] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the enzymes of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0087] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0088] Transcription of the DNA encoding the enzymes of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0089] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated enzyme. Optionally, the heterologoussequence can encode a fusion enzyme including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

[0090] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0091] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0092] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0093] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0094] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well known to those skilled in the art.

[0095] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0096] The enzyme can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0097] The enzymes of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the enzymes of the present invention may beglycosylated or may be non-glycosylated. Enzymes of the invention may ormay not also include an initial methionine amino acid residue.

[0098] Esterases are a group of key enzymes in the metabolism of fatsand are found in all organisms from microbes to mammals. In thehydrolysis reaction, an ester group is hydrolysed to an organic acid andan alcohol.

[0099] Esterases enantiomerically differentiate dicarboxylic diestersand diacetates of diols. Using the approach disclosed in a commonlyassigned, copending provisional application Serial No. 60/008,316, filedon Dec. 7, 1995 and entitled “Combinatorial Enzyme Development,” thedisclosure of which is incorporated herein by reference in its entirety,one could convert the enantiospecificity of the esterase. Further, thethermostable esterases are believed to have superior stability at highertemperatures and in organic solvents. Thus, they are better suited foruse in rigorous production procees which require robust catalysts.

[0100] There are a number of industrial and scientific applications foresterases, such as those of the present invention, including:

[0101] 1) Esterases are useful in the dairy industry as ripeningstarters for cheeses, such as the Swiss-type cheeses:

[0102] 2) Esterases are useful in the pulp and paper industry for ligninremoval from cellulose pulps, for lignin solubilization by cleaving theester linkages between aromatic acids and lignin and between lignin andhemicelluloses, and for disruption of cell wall structure when used incombination with xylanase and other xylan-degrading enzymes inbiopulping and biobleaching of pulps;

[0103] 3) Esterases are useful in the synthesis of carbohydratederivatives, such as sugar derivatives;

[0104] 4) Esterases are useful, when combined with xylanases andcellulases, in the conversion of lignocellulosic wastes to fermentablesugars for producing a variety of chemicals and fuels;

[0105] 5) Esterases are useful as research reagents in studies on plantcell wall structure, particularly the nature of covalent bonds betweenlignin and carbohydrate polymers in the cell wall matrix;

[0106] 6) Esterases are also useful as research reagents in studies onmechanisms related to disease resistance in plants and the process oforganic matter decomposition; and

[0107] 7) Esterases are useful in selection of plants bred forproduction of highly digestible animal feeds, particularly for ruminantanimals.

[0108] Antibodies generated against the enzymes corresponding to asequence of the present invention can be obtained by direct injection ofthe enzymes into an animal or by administering the enzymes to an animal,preferably a nonhuman. The antibody so obtained will then bind theenzymes itself. In this manner, even a sequence encoding only a fragmentof the enzymes can be used to generate antibodies binding the wholenative enzymes. Such antibodies can then be used to isolate the enzymefrom cells expressing that enzyme.

[0109] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,Nature, 256:495-497, 1975), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4:72, 1983), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96, 1985).

[0110] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic enzyme products of this invention. Also,transgenic mice may be used to express humanized antibodies toimmunogenic enzyme products of this invention.

[0111] Antibodies generated against an enzyme of the present inventionmay be used in screening for similar enzymes from other organisms andsamples. Such screening techniques are known in the art, for example,one such screening assay is described in Sambrook et al., MolecularCloning: A Laboratory Manual (2d Ed.), Cold Spring Harbor Laboratory,Section 12.21-12.28 (1989) which is hereby incorporated by reference inits entirety.

[0112] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0113] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0114] “Plasmids” are designated by a lower case “p” preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0115] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0116] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel et al., Nucleic AcidsRes., 8:4057 (1980).

[0117] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0118] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0119] Unless otherwise stated, transformation was performed asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual (2dEd.), Cold Spring Harbor Press (1989).

EXAMPLE 1 Bacterial Expression and Purification of Esterases

[0120] DNA encoding the enzymes of the present invention, SEQ ID NOS:33through 42, were initially amplified from a pBluescript vectorcontaining the DNA by the PCR technique using the primers noted herein.The amplified sequences were then inserted into the respective PQEvector listed beneath the primer sequences, and the enzyme was expressedaccording to the protocols set forth herein. The 5′ and 3′ primersequences for the respective genes are as follows: Staphylothermusmarinus F1-12LC 5′CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGTCTTTA AACAAGCACTCT 3′CGGAAGATCT CTATCGTTTA GTGTATGATT T vector: pQET PyrodictiumTAG11-17LC 5′CCGAGAATTC ATTAAAGAGG AGAAATTAAC EcoRI TATGAAACTCCTTGAGCCCA CA 3′CGGAAGATCT CGCCGGTACA CCATCAGCCA C BglII vector: pQETArchaeoglobus venificus SNP6-24LC 5′CCGAGAATTC ATTAAAGAGG AGAAATTAACTATGCCATAT GTTAGGAATG GT 3′CGGAGGTACC TTAGAACTGT GCTGAAGAAA TAAATTCGTCCATTGCTCT 3′CGGAGGTACC TTAGAACTGT GCTGAAGAAA TAAATTCGTC CATTGCTCTA TTAvector: pQET Aquifex pyraphilus-28LC 5′CCGAGAATTC ATTAAAGAGG AGAAATTAACTATGAGATTG AGGAAATTTG AAG 3′CGGAGGTACC CTATTCAGAA AGTACCTCTA A vector:pQET M11TL-29LC 5′CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGTTTAAT ATCAATGTCTTT 3′CGGAAGATCT TTAAGGATTT TCCCTGGGTA G vector: pQET ThermococcusCL-2-30LC 5′CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGGAGGTT TACAAGGCCA AA3′CGGAGGTACC TTATTGAGCC GAAGAGTACG A vector: pQET Aquifex VF5-34LC5′CCGAGAATTC ATTAAAGAGG AGAAATTAAC EcoRI TATGATTGGC AATTTGAAAT TGA3′CGGAGGTACC TTAAAGTGCT CTCATATCCC C KpnI vector: pQET Teredinibacter42L 5′CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGCCAGCT AATGACTCAC CC3′CGGAAGATCT TCAACAGGCT CCAAATAATT TC (without His-tag) 3′CGGAAGATCTACAGGCTCCA AATAATTTC (with His-tag) vector: pQE12 Archaeoglobus fulgidusVC16-16MC 5′CCGAGAATTC ATTAAAGAGG AGAAATTAAC EcoR1 TATGCTTGAT ATGCCAATCGAC 3′CGGAGGTACC CTAGTCGAAG ACAAGAAGAG C Kpn1 vector: pQET Sulfolabussolfataricus P1-8LC 5′CCGAGAATTC ATTAAAGAGG AGAAATTAAC EcoR1 TATGCCCCAGGATCCTAGAA TT 3′CGGAGGTACC TTAAATTTTA TCATAAAATA C Kpn1 vector: pQET

[0121] The restriction enzyme sites indicated correspond to therestriction enzyme sites on the bacterial expression vector indicatedfor the respective gene (Qiagen, Inc. Chatsworth, Calif.). The pQEvector encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.

[0122] The pQE vector was digested with the restriction enzymesindicated. The amplified sequences were ligated into the respective pQEvector and inserted in frame with the sequence encoding for the RBS. Theligation mixture was then used to transform the E. coli strain M15/pREP4(Qiagen, Inc.) by electroporation. M15/pREP4 contains multiple copies ofthe plasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)). Transformants were identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and confirmed by restrictionanalysis. Clones containing the desired constructs were grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture was used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/0 leading to increased gene expression. Cells were grownan extra 3 to 4 hours. Cells were then harvested by centrifugation.

[0123] The primer sequences set out above may also be employed toisolate the target gene from the deposited material by hybridizationtechniques described above.

EXAMPLE 2 Isolation of a Selected Clone from the Deposited GenomicClones

[0124] The two oligonucleotide primers corresponding to the gene ofinterest are used to amplify the gene from the deposited material. Apolymerase chain reaction is carried out in 25 μl of reaction mixturewith 0.1 μg of the DNA of the gene of interest. The reaction mixture is1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP,dTTP, 25 pmol of each primer and 1.25 Unit of Taq polymerase. Thirtycycles of PCR (denaturation at 94° C. for 1 min: annealing at 55° C. for1 min; elongation at 72° C. for 1 min) are performed with thePerkin-Elmer Cetus 9600 thermal cycler. The amplified product isanalyzed by agarose gel electrophoresis and the DNA band with expectedmolecular weight is excised and purified. The PCR product is verified tobe the gene of interest by subcloning and sequencing the DNA product.

EXAMPLE 3 Production of the Expression Gene Bank

[0125] Colonies containing pBluescript plasmids with random inserts fromthe organisms M11TL, Thermococcus GU5L5, and Teredinibacter wereobtained according to the method of Hay and Short, Strategies, 5:16,1992.

EXAMPLE 4 Screening for Lipase/Esterase Activity

[0126] The resulting colonies were picked with sterile toothpicks andused to singly inoculate each of the wells of 96-well microtiter plates.The wells contained 250 μL of LB media with 100 μg/mL ampicillin, 80μg/mL methicillin, and 10% v/v glycerol (LB Amp/Meth, glycerol). Thecells were grown overnight at 37° C. without shaking. This constitutedgeneration of the “Source GeneBank.” Each well of the Source GeneBankthus contained a stock culture of E. coli cells, each of which containeda pBluescript with a unique DNA insert.

[0127] The plates of the Source GeneBank were used to multiply inoculatea single plate (the “Condensed Plate”) containing in each well 200 μL ofLB Amp/Meth, glycerol. This step was performed using the High DensityReplicating Tool (HDRT) of the Beckman Biomek with a 1% bleach, water,isopropanol, air-dry sterilization cycle in between each inoculation.Each well of the Condensed Plate thus contained 10 to 12 differentpBluescript clones from each of the source library plates. The CondensedPlate was grown for 16 hours at 37° C. and then used to inoculate twowhite 96-well Polyfiltronics microtiter daughter plates containing ineach well 250 μL of LB Amp/Meth (no glycerol). The original condensedplate was put in storage −80° C. The two condensed daughter plates wereincubated at 37° C. for 18 hours.

[0128] The short chain esterase ‘600 μM substrate stock solution’ wasprepared as follows: 25 mg of each of the following compounds wasdissolved in the appropriate volume of DMSO to yield a 25.2 mM solution.The compounds used were 4-methylumbelliferyl proprionoate,4-methylumbelliferyl butyrate, and 4-methylumbelliferyl heptanoate. Twohundred fifty microliters of each DMSO solution was added to ca 9 mL of50 mM, pH 7.5 Hepes buffer which contained 0.6% of Triton X-100 and 0.6mg per mL of dodecyl maltoside (Anatrace). The volume was taken to 10.5mL with the above Hepes buffer to yield a slightly cloudy suspension.

[0129] The long chain ‘600 μM substrate stock solution’ was prepared asfollows: 25 mg of each of the following compounds was dissolved in DMSOto 25.2 mM as above. The compounds used were 4-methylumbelliferylelaidate, 4-methylumbelliferyl palmitate, 4-methylumbelliferyl oleate,and 4-methylumbelliferyl stearate. All required brief warming in a 70°C. bath to achieve dissolution. Two hundred fifty microliters of eachDMSO solution was added to the Hepes buffer and diluted to 10.5 mL asabove. All seven umbelliferones were obtained from Sigma Chemical Co.

[0130] Fifty μL of the long chain esterase or short chain esterase ‘600μM substrate stock solution’ was added to each of the wells of a whitecondensed plate using the Biomek to yield a final concentration ofsubstrate of about 100 μM. The fluorescence values were recorded(excitation=326 nm, emission=450 nm) on a plate-reading fluorometerimmediately after addition of the substrate. The plate was incubated at70° C. for 60 minutes in the case of the long chain substrates, and 30minutes at RT in the case of the short chain substrates. Thefluorescence values were recorded again. The initial and finalfluorescence values were compared to determine if an active clone waspresent.

EXAMPLE 5 Isolation and Purification of the Active Clone

[0131] To isolate the individual clone which carried the activity, theSource GeneBank plates were thawed and the individual wells used tosingly inoculate a new plate containing LB Amp/Meth. As above, the platewas incubated at 37° C. to grow the cells, 50 μL of 600 μM substratestock solution was added using the Biomek and the fluorescence wasdetermined. Once the active well from the source plate was identified,cells from this active well were streaked on agar with LB/Amp/Meth andgrown overnight at 37° C. to obtain single colonies. Eight singlecolonies were picked with a sterile toothpick and used to singlyinoculate the wells of a 96-well microtiter plate. The wells contained250 μL of LB Amp/Meth. The cells were grown overnight at 37° C. withoutshaking. A 200 μL aliquot was removed from each well and assayed withthe appropriate long or short chain substrates as above. The most activeclone was identified and the remaining 50 μL of culture was used tostreak an agar plate with LB/Amp/Meth. Eight single colonies werepicked, grown and assayed as above. The most active clone was used toinoculate 3 mL cultures of LB/Amp/Meth, which were grown overnight. Theplasmid DNA was isolated from the cultures and utilized for sequencing.

[0132] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

What is claimed is:
 1. An enzyme comprising a member selected from thegroup consisting of an enzyme comprising an amino acid sequence which isat least 70% identical to the amino acid sequence set forth in SEQ IDNOS:33-42